FastLane Cell Multiplex Kits accelerate and streamline real-time RT-PCR and are ideal for experiments requiring rapid high-throughput gene expression analysis. The kits provide a set of buffers for preparing FastLane lysates from cultured cells. In addition to lysing cells, the buffers also stabilize cellular RNA and eliminate genomic DNA. FastLane lysates are used directly in multiplex, real-time one-step RT-PCR (with or without ROX passive reference dye). A variety of other detection formats are also available, including detection with SYBR Green or sequence-specific probes.

Multiplex, real-time one-step RT-PCR analysis of the expression of NFκB1 (nuclear transcription factor) and GAPDH (endogenous control) using the FastLane Kit gave reproducible CT values from a 96-well plate of HeLa cells.

Performance

With FastLane Cell Multiplex Kits, multiplex analysis in 96- and 384-well formats is highly reproducible (see figure "Highly reproducible analysis of a 96-well plate"). The kits are ideal for rapid, high-throughput gene expression analysis, such as validation of siRNA-mediated gene knockdown (see figure "Reliable validation of gene silencing"). Target and control genes are amplified in the same reaction with similar, high PCR efficiencies, enabling relative quantification by the ΔΔCT method.

Principle

FastLane Cell Multiplex Kits use FastLane technology to prepare cell lysates for real-time one-step RT-PCR directly from cultured cells. The kits accelerate and streamline real-time PCR analysis of cultured cells by eliminating the need for RNA purification and are ideal for rapid, high-throughput gene expression analysis, such as validation of siRNA-mediated gene knockdown.

Unlike other methods, FastLane Cell Multiplex Kits deliver success in multiplex, real-time one-step RT-PCR at the first attempt (see figure "Multiplex analysis with no optimization steps") due to an optimized master mix containing a novel RT-PCR buffer and a hot-start DNA polymerase. Two kit formats are available: the FastLane Cell Multiplex Kit for cyclers that require ROX dye for fluorescence normalization, and the FastLane Cell Multiplex NR Kit for all other cyclers.

Procedure

With FastLane Cell Multiplex Kits, lysates are obtained in just 12 minutes in a 3-step procedure: cell wash; cell lysis with integrated RNA stabilization and genomic DNA elimination; and lysate pretreatment. After pretreatment, lysates are ready to use in real-time one-step RT-PCR.

Each kit contains sufficient reagents for cell lysis in two 96-well cell culture plates and subsequent real-time RT-PCR (50 µl PCR volume). Each kit can also be used to analyze a single 384-well cell culture plate if the PCR volume is reduced to 20 µl; if a 50 µl PCR volume is desired, an additional QuantiTect Multiplex RT-PCR Kit (200) or QuantiTect Multiplex RT-PCR NR Kit (200) needs to be purchased.